本文選題：頸動脈體 + 竇神經(jīng)��； 參考：《第四軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 應(yīng)激是機體在面對環(huán)境中不利因素時,實際或認知上的要求與適應(yīng)和應(yīng)付能力之間的不平衡所導(dǎo)致的身心緊張狀態(tài)及其反應(yīng)。對應(yīng)激環(huán)路和調(diào)控機制的研究對闡明多種疾病的發(fā)病機理及尋找針對應(yīng)激(包括軍事應(yīng)激)的應(yīng)對措施有重要的意義。應(yīng)激反應(yīng)實質(zhì)上是神經(jīng)-免疫-內(nèi)分泌網(wǎng)絡(luò)中各系統(tǒng)共同反應(yīng)的綜合體現(xiàn)。隨著研究的不斷深入,人們逐漸認識到中樞神經(jīng)系統(tǒng)(central nervous system, CNS)、免疫系統(tǒng)、內(nèi)分泌系統(tǒng)并不是各自為政、獨立存在的系統(tǒng),它們之間存在著密切的相互聯(lián)系和調(diào)控。一般認為,CNS通過下丘腦-垂體-腎上腺軸、下丘腦-垂體-性腺軸以及植物性神經(jīng)系統(tǒng)等傳出通路調(diào)控和影響免疫、內(nèi)分泌系統(tǒng)的功能1-4。內(nèi)分泌系統(tǒng)也通過一些靶腺激素的反饋機制影響CNS的功能。但是,免疫系統(tǒng)是通過何種途徑影響CNS功能的?外周血液中的細胞因子是通過怎樣的方式將“信息”傳遞給CNS的?一直是個未解之謎。 在機體遭受病原體刺激早期,外周免疫系統(tǒng)中的細胞即迅速作出反應(yīng),釋放出多種促炎性細胞因子,如:IL-1β、TNFα、IL-6、IFNγ等。一些研究發(fā)現(xiàn),在免疫應(yīng)激或者感染的情況下,IL-1β是中樞性發(fā)熱反應(yīng)的始動因子。因此,IL-1β可能是在外周免疫系統(tǒng)和CNS之間發(fā)揮重要“信使”作用的分子之一3,5。但由于IL-1β分子量較大,且具有親水性的特點,因而幾乎不能通過血腦屏障(blood brain barrier,BBB)6。有人認為,在腦室周圍腦組織中存在一些BBB缺如的區(qū)域,外周循環(huán)中的IL-1β可能通過這些區(qū)域進入CNS;還有人認為細胞因子可能在與腦血管內(nèi)皮細胞上的受體結(jié)合后,產(chǎn)生一些下游分子(例如前列腺素E2等),將外周免疫信號傳遞入腦4,7。然而,資料表明通過這些途徑入腦的細胞因子量很少8。最近,外周感覺神經(jīng),尤其是迷走神經(jīng)的感覺傳入纖維在“免疫-腦”通信傳入途徑中的作用,逐漸引起人們廣泛的關(guān)注9,10。一些研究者發(fā)現(xiàn),迷走神經(jīng)一些分支的終末或者神經(jīng)末梢旁的多巴胺能細胞(這些細胞常聚集成團稱為迷走神經(jīng)旁神經(jīng)節(jié),受迷走神經(jīng)傳入纖維支配)具有IL-1β等細胞因子的結(jié)合位點11;推測細胞因子可能通過直接刺激迷走神經(jīng)末梢,或通過旁神經(jīng)節(jié)細胞的換能,引起迷走神經(jīng)傳入纖維放電頻率的改變,向CNS傳遞外周免疫刺激信號12。 哺乳動物的頸動脈體(carotid body, CB)是一個位于頸動脈血管分叉處的血運極其豐富的器官。傳統(tǒng)觀念認為,CB是重要的外周化學(xué)感受器,可以非常精確、靈敏地感受動脈血中的氧分壓(PO2)、二氧化碳分壓(PCO2)、酸堿度(pH)和滲透壓等的變化13。最新的研究發(fā)現(xiàn),CB還可以感受外周循環(huán)中葡萄糖濃度的變化14。這些發(fā)現(xiàn)不斷地揭示了CB化學(xué)感受功能的多樣性和復(fù)雜性。 根據(jù)解剖學(xué)的分類,CB也屬于旁神經(jīng)節(jié)。同迷走神經(jīng)旁神經(jīng)節(jié)一樣15,CB也是由主細胞(球細胞,I型細胞)和支持細胞(II型細胞)組成;其球細胞是多巴胺能細胞,可以合成、儲存、分泌兒茶酚胺類遞質(zhì)。我們實驗室前期的工作發(fā)現(xiàn),在正常大鼠的球細胞上有強烈的IL-1受體I型(IL-1RI)16和IL-6受體17表達。因此,我們推測:CB除了傳統(tǒng)的化學(xué)感受功能外,還可以感受外周循環(huán)中的免疫刺激信號(如:IL-1、IL-6等),并通過其傳入神經(jīng)—竇神經(jīng)(carotid sinus nerve, CSN)的放電頻率改變向中樞神經(jīng)系統(tǒng)傳遞信息。 本研究的主要目的是驗證上述假說。我們先在細胞特性和CB球細胞非常相似的PC12細胞進行了初步觀察。PC12細胞是源自于大鼠腎上腺嗜鉻細胞腫瘤的細胞系。同嗜鉻細胞一樣,PC12細胞也可以合成、儲存、分泌兒茶酚胺類遞質(zhì)。另外,PC12細胞在NGF誘導(dǎo)下可以分化成具有交感神經(jīng)元特性的細胞18,因此,PC12細胞作為一種細胞模型,被廣泛用于神經(jīng)元的藥理、生理學(xué)研究19-21。近年來的研究發(fā)現(xiàn), PC12細胞同CB的球細胞具有很多類似之處。例如:PC12細胞和CB的球細胞膜上都有氧敏感的鉀通道22,23,PC12細胞也可以感受低氧、低pH等刺激。在組織學(xué)上,腎上腺嗜鉻細胞和CB細胞都起源于神經(jīng)嵴,它們都可以合成并分泌多巴胺(dopamine, DA)、乙酰膽堿(acetylcholine, ACh)等神經(jīng)遞質(zhì)13,24。大量的實驗證據(jù)發(fā)現(xiàn),PC12細胞和CB球細胞具有很多生理學(xué)、藥理學(xué)的共性25-27。因此,PC12細胞也被廣泛用作外周化學(xué)感受性細胞(如CB球細胞)功能研究的細胞模型和工具細胞。 在本研究中,我們綜合應(yīng)用免疫細胞化學(xué)技術(shù)、膜片鉗技術(shù)、鈣成像技術(shù)、在體胞外記錄技術(shù)以及離體和在體的電化學(xué)分析檢測等技術(shù),首先用PC12細胞模型研究了外源性IL-1β刺激對PC12細胞電生理特性以及細胞內(nèi)游離鈣離子濃度([Ca2+]i)的影響。然后,從培養(yǎng)細胞、組織切片以及在體水平,分別研究了外源性IL-1β刺激對大鼠CB球細胞的電生理特性及其傳入神經(jīng)—CSN放電頻率的影響。另外為了下一步工作的開展,我們還研究了CB的中最重要的神經(jīng)遞質(zhì)之一——DA在急性缺氧誘發(fā)放電中的作用。 結(jié)果簡要歸納如下: (1)免疫細胞化學(xué)和蛋白質(zhì)印跡分析結(jié)果發(fā)現(xiàn),在PC12細胞的胞膜和胞核上均有IL-1RI的分布。電生理結(jié)果表明,IL-1β可以通過和胞膜上特異的受體結(jié)合,濃度依賴性地抑制PC12細胞上外向性電壓依賴的鉀電流(IK)的幅度。而且,IL-1β可以引起IK的失活曲線左移,但不影響其激活曲線。另外,IL-1β可以引起PC12細胞膜超極化,并可引起[Ca~(2+)]i迅速升高。 (2)全細胞膜片鉗記錄和鈣成像結(jié)果顯示,胞外給予IL-1β可以顯著地抑制CB球細胞外向型鉀電流的幅度,并可導(dǎo)致[Ca~(2+)]i迅速升高。在體的細胞記錄結(jié)果顯示,藥理濃度的IL-1β刺激可以使麻醉大鼠的CSN放電活性增強。進一步的研究提示,CB球細胞感受IL-1β刺激的突觸傳遞過程可能有ATP的參與,而DA在這一過程中可能不起主要作用。 (3)與以往的離體實驗結(jié)果相反,在體情況下實驗性急性缺氧引起CB內(nèi)兒茶酚胺類遞質(zhì)的釋放急劇降低,且這種效應(yīng)可以被切斷CB的傳入神經(jīng)CSN所消除或者減弱。進一步的研究發(fā)現(xiàn):①在in vivo記錄情況下,CB局部給予DA可以抑制CSN的放電活性,這與以往文獻報道的結(jié)果一致28-31;②CB局部給予D2受體的阻斷劑haloperidol (Hal)可以引起CSN自發(fā)放電的頻率升高,而局部給予D1受體阻斷劑SCH23390則未見CSN放電頻率的明顯改變;③DA可能通過D1或者D2受體引起CB球細胞內(nèi)吞作用增強。④腺苷(adenosine)可引起缺氧誘導(dǎo)的離體CB釋放CAs減少。 主要結(jié)論: (1)PC12細胞對胞外IL-1β刺激起反應(yīng); (2)CB及其傳入神經(jīng)CSN對外源性IL-1β的直接刺激起反應(yīng),提示其可能具有感受外周免疫刺激(如IL-1β)的能力,可能是“免疫-腦”通信的感受器之一; (3)缺氧時, CB中胞外DA量通過腺苷的調(diào)節(jié)作用而減少,從而增加了CSN的放電頻率。推測CB可能利用DA的這種“推-挽”機制而改變CSN的放電,將外周的缺氧信號傳向中樞神經(jīng)系統(tǒng)。
[Abstract]:Stress is the physical and mental state and reaction of the body in the face of adverse factors in the environment, the actual or cognitive requirements and the imbalance between adaptation and coping ability. The study of the stress loop and regulatory mechanism is important to elucidate the pathogenesis of various diseases and to find the response to stress (including military stress). The stress response is essentially a comprehensive reflection of the common reactions of the systems in the neural immuno endocrine network. As the research progressively deepened, people gradually realized that the central nervous system (central nervous system, CNS), the immune system, and the endocrine system were not independent and independent systems, and there was a secret between them. It is generally believed that CNS regulates and affects immunization through the hypothalamus pituitary adrenal axis, the hypothalamus pituitary - gonadal axis and the plant nerve system. The function of the endocrine system of the endocrine system also affects the function of CNS through the feedback mechanism of some target adenoids. However, the immune system is through. Which way to influence the function of CNS? How can cytokines be transferred to CNS in peripheral blood?
In the early stage of the pathogen stimulation, the cells in the peripheral immune system respond quickly and release a variety of proinflammatory cytokines, such as IL-1 beta, TNF alpha, IL-6, IFN gamma, etc. some studies have found that, in the case of immune stress or infection, IL-1 beta is the starting factor of the central fever reaction. Therefore, IL-1 beta may be in the peripheral immune system. 3,5., one of the most important "messenger" molecules between the system and the CNS, has been found to be almost impossible to pass through the blood brain barrier (blood brain barrier, BBB) 6. because of its large molecular weight and hydrophilicity. There are some BBB absent regions in the brain tissue around the ventricle, and the IL-1 beta in the peripheral circulation may pass through this Some regions enter CNS; others think that cytokines may be associated with receptors on cerebral vascular endothelial cells and produce some downstream molecules, such as prostaglandin E2, to transfer peripheral immune signals into the brain 4,7.. The role of nerve sensory afferent fibers in the "immuno brain" communication pathway has gradually aroused widespread concern in 9,10. researchers. Some researchers found that the terminals of some branches of the vagus nerve, or the dopaminergic cells near the nerve endings, are called the paraplastic ganglia, which are afferent fibers of the vagus nerve. The binding site of IL-1 beta and other cytokines is 11. It is speculated that cytokines may directly stimulate the endings of the vagus nerve or through the transfer of paraganglionic cells, causing the change of the frequency of the afferent fibers of the vagus nerve and the transmission of the peripheral immune stimulation signal to CNS by 12..
The carotid body (CB) in mammals is an extremely rich organ in the bifurcation of the carotid artery. The traditional idea is that CB is an important peripheral chemoreceptor and can be very accurate and sensitive to the oxygen partial pressure (PO2), the two carbon oxide pressure (PCO2), the pH (pH) and osmotic pressure in the arterial blood. 13. The latest research has found that CB can also feel the changes in glucose concentration in the peripheral circulation 14.. These findings continue to reveal the diversity and complexity of CB chemoreceptor function.
According to the anatomical classification, CB also belongs to the paraplastic ganglion. Like the paraceal ganglion, 15, CB is also composed of main cells (ball cells, I cells) and support cells (II cells); the ball cells are dopaminergic cells that can synthesize, store and secrete catechol amine transmitters. Our work in the early laboratory found that in normal rats There is a strong expression of IL-1 receptor I (IL-1RI) 16 and IL-6 receptor 17 on the ball cells. Therefore, we speculate that, in addition to the traditional chemoreceptor function, CB can also feel the immune stimulation signals in the peripheral circulation (such as IL-1, IL-6, etc.), and change the frequency of the discharge to the central nerve through the frequency of its afferent nerve - sinus nerve (carotid sinus nerve, CSN). The system passes information.
The main purpose of this study is to verify the hypothesis. First, we preliminarily observed that.PC12 cells were derived from rat adrenal chromaffin cells in PC12 cells with very similar cell characteristics and CB cells. Like chromaffin cells, PC12 cells can also be synthesized, stored, and secreted by catecholamine transmitters. In addition, PC12 cells are secreted. NGF induced cells can be differentiated into 18 cells with the characteristics of sympathetic neurons. Therefore, PC12 cells are widely used as a cell model and are widely used in the pharmacological activities of neurons. In recent years, the study of 19-21. found that PC12 cells have many similarities with CB cells. For example, PC12 cells and the membrane of CB are sensitive to oxygen. The potassium channel 22,23, PC12 cells also can feel hypoxic, low pH and other stimuli. Histologically, adrenal chromaffin and CB cells are all derived from the neural crest, and they can synthesize and secrete dopamine (dopamine, DA), acetylcholine (acetylcholine, ACh) and other neurotransmitter 13,24. experimental evidence found, PC12 cells and CB spherical cells. As a result of many physiological and pharmacological generalities, 25-27., PC12 cells are also widely used as cell models and tool cells for the study of peripheral chemoreceptor cells (such as CB spherocytes).
In this study, we used immunocytochemical techniques, patch clamp techniques, calcium imaging techniques, extracellular recording techniques, and electrochemical detection techniques in vitro and in vivo. The electrophysiological characteristics of exogenous IL-1 beta stimulation and intracellular free calcium concentration ([Ca2+]i) in PC12 cells were first studied by PC12 cell model. Then, the effects of exogenous IL-1 beta stimulation on the electrophysiological characteristics of CB cells and the frequency of afferent nerve CSN discharge were studied from cultured cells, tissue sections and at the body level. In addition, in order to carry out the next step, we also studied one of the most important neurotransmitters in CB, DA in acute deficiency. The role of oxygen induced discharge.
The results are summarized as follows:
(1) the results of immunocytochemistry and Western blot analysis showed that there was a distribution of IL-1RI in the cell membrane and nucleus of PC12 cells. Electrophysiological results showed that IL-1 beta could bind to the specific receptors on the membrane and inhibit the extrovert voltage of PC12 cells dependent on the amplitude of the potassium current (IK) of the PC12 cells. Moreover, IL-1 beta could cause IK. The inactivation curve shifted to the left, but did not affect its activation curve. In addition, IL-1 beta could induce hyperpolarization of PC12 cell membrane and cause rapid rise of [Ca~ (2+)]i.
(2) whole cell patch clamp recording and calcium imaging results showed that extracellular given IL-1 beta could significantly inhibit the amplitude of extrovertic potassium current of CB cells and lead to a rapid increase in [Ca~ (2+)]i. The results of cell recording in vivo showed that IL-1 beta stimulation of pharmacological concentration could enhance the activity of CSN discharge in anesthetized rats. Further research suggests CB The involvement of ATP in the synaptic transmission of IL-1 beta stimulated by spheroid cells may not play a major role in this process.
(3) contrary to the previous experimental results, the release of catecholamine transmitters in CB is dramatically reduced in the experimental acute hypoxia, and this effect can be eliminated or weakened by the afferent CSN of the CB. Further studies have found that in the in vivo record, the partial administration of CB to DA can inhibit the discharge of CSN. Sex, this is in agreement with the results reported in the previous literature. (2) haloperidol (Hal), blocking agent haloperidol (Hal) locally given to CB receptor, can cause an increase in the frequency of spontaneous discharge of CSN, while local administration of D1 receptor blocker SCH23390 has not seen a significant change in the frequency of CSN discharge; (3) DA may increase the endocytosis by D1 or D2 receptors. 4 Adenosine (adenosine) can induce hypoxia induced CAs release from CB release in vitro.
The main conclusions are as follows:
(1) PC12 cells responded to extracellular IL-1 beta stimulation.
(2) CB and its afferent nerve CSN react with direct stimulation of external IL-1 beta, suggesting that it may have the ability to feel peripheral immune stimulation (such as IL-1 beta), which may be one of the receptors for "immuno brain" communication.
(3) when hypoxia, the amount of extracellular DA in CB is reduced by the regulation of adenosine, thus increasing the discharge frequency of CSN. It is presumed that CB may use the "push pull" mechanism of DA to change the discharge of CSN and transmit the peripheral anoxic signal to the central nervous system.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R363

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